Coronaviruses, a genus in the family Coronoviridae, are large, enveloped RNA viruses that cause highly prevalent diseases in humans and domestic animals. Coronavirus particles are irregularly-shaped, 60-220 nm in diameter, with an outer envelope bearing distinctive, “club-shaped” peplomers. This “crown-like” appearance gives the family its name. Coronaviruses have the largest genomes of all RNA viruses and replicate by a unique mechanism which results in a high frequency of recombination. Virions mature by budding at intracellular membranes and infection with some coronaviruses induces cell fusion.
Most human coronaviruses (HcoVs) do not grow in cultured cells, therefore relatively little is known about them, but two strains (229E and OC43) grow in some cell lines and have been used as a model. Replication is slow compared to other enveloped viruses. Viral entry occurs via endocytosis and membrane fusion (probably mediated by E2) and replication occurs in the cytoplasm.
Initially, the 5′ 20 kb of the (+)sense genome is translated to produce a viral polymerase, which is believed to produce a full-length (−)sense strand which, in turn, is used as a template to produce mRNA as a “nested set” of transcripts, all with an identical 5′ non-translated leader sequence of 72 nucleotides and coincident 3′ polyadenylated ends. Each mRNA is monocistronic, the genes at the 5′ end being translated from the longest mRNA. These unusual cytoplasmic structures are produced not by splicing (post-transcriptional modification) but by the polymerase during transcription.
Coronaviruses infect a variety of mammals and birds. The exact number of human isolates is not known as many cannot be grown in culture. In humans, they cause: respiratory infections (common), including Severe Acute Respiratory Syndrome (SARS), and enteric infections.
Coronaviruses are transmitted by aerosols of respiratory secretions, by the fecal-oral route, and by mechanical transmission. Most virus growth occurs in epithelial cells. Occasionally the liver, kidneys, heart or eyes may be infected, as well as other cell types such as macrophages. In cold-type respiratory infections, growth appears to be localized to the epithelium of the upper respiratory tract, but there is currently no adequate animal model for the human respiratory coronaviruses. Clinically, most infections cause a mild, self-limited disease (classical “cold” or upset stomach), but there may be rare neurological complications.
Coronavirus infection is very common and occurs worldwide. The incidence of infection is strongly seasonal, with the greatest incidence in children in winter. Adult infections are less common. The number of coronavirus serotypes and the extent of antigenic variation are unknown. Re-infections appear to occur throughout life, implying multiple serotypes (at least four are known) and/or antigenic variation, hence the prospects for immunization appear bleak.
SARS (Severe Acute Respiratory Syndrome) is a newly-recognized type of viral pneumonia, with symptoms including fever, a dry cough, dyspnea (shortness of breath), headache, and hypoxemia (low blood oxygen concentration). Typical laboratory findings include lymphopenia (reduced lymphocyte numbers) and mildly elevated aminotransferase levels (indicating liver damage). Death may result from progressive respiratory failure due to alveolar damage.
The outbreak is believed to have originated in February 2003 in the Guangdong province of China. After initial reports that a paramyxovirus was responsible, researchers now believe SARS to causually-linked with a type of novel coronavirus with some unusual properties. For example, the SARS virus can be grown in Vero cells (a primate fibroblast cell line)—a novel property for HCoVs, most of which cannot be cultivated. In these cells, virus infection results in a cytopathic effect, and budding of coronavirus-like particles from the endoplasmic reticulum within infected cells.
Amplification of short regions of the polymerase gene, (the most strongly conserved part of the coronavirus genome) by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing revealed that the currently evaluated examples of the SARS virus are of a novel coronavirus which has not previously been present in human populations.
Different isolates of coronaviruses that have been causally linked to SARS have been independently sequenced by BCCA Genome Sciences Center, Vancouver, Canada; the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences/Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, China; the Centers for Disease Control and Prevention (CDC), Atlanta; the Chinese University of Hong Kong; and the University of Hong Kong. As new SARS-linked coronavirus samples are obtained and sequenced, and as the initial SARS coronaviruses mutate, other coronavirus sequences causally-linked to SARS will emerge.
While the SARS epidemic is still at the early stages, Ruan et al have identified a number of variations in existing SARS CoV isolates that suggest the emergence of new genotypes (Y. Ruan et al., Lancet, May 9, (2003)). This phenomenon is likely to continue if SARS CoV passes through the human population and will have a detrimental impact on detection and treatment. Additional primers that flank regions of high variability could be valuable in epidemiological tracking of strain variants. Moreover, as loci important to virulence become identified, primers that flank these locations could provide valuable information.
Diagnostic tests are now available, but all have limitations as tools for bringing this outbreak quickly under control. An ELISA test detects antibodies reliably but only from about day 20 after the onset of clinical symptoms. It therefore cannot be used to detect cases at an early stage prior to spread of the infection to others. The second test, an immunofluorescence assay (IFA), detects antibodies reliably as of day 10 of infection. It shares the defect of the ELISA test in that test subjects have become infective prior to IFA-based diagnosis. Moreover, the IFA test is a demanding and comparatively slow test that requires the growth of virus in cell culture. The third test is a polymerase chain reaction (PCR) molecular test for detection of SARS virus genetic material is useful in the early stages of infection but undesirably produces false-negatives. Thus the PCR test may fail to detect persons who actually carry the virus, even in conjunction with clinical diagnostic evaluation, creating a dangerous sense of false security in the face of a potential epidemic of a virus that is known to spread easily in close person-to-person contact (WHO. Severe acute respiratory syndrome (SARS). Wkly Epidemiol. Rec. 2003, 78, 121-122).
Nucleic acid tests for infectious diseases are largely based upon amplifications using primers and probes designed to detect specific bioagents. Because prior knowledge of nucleic acid sequence information is required to develop these tests they are not able to identify unanticipated, newly emergent, or previously unknown infectious bioagents. Thus, the initial discovery of infectious bioagents still relies largely on culture and microscopy, which were as important in the recent identification of the SARS coronavirus as they were in the discovery of the human immunodeficiency virus two decades ago.
An alternative to single-agent tests is to do broad-range consensus priming of a gene target conserved across groups of bioagents. Broad-range priming has the potential to generate amplification products across entire genera, families, or, as with bacteria, an entire domain of life. This strategy has been successfully employed using consensus 16S ribosomal RNA primers for determining bacterial diversity, both in environmental samples (T. M. Schmidt, T. M., DeLong, E. F., Pace, N. R. J. Bact. 173, 4371-4378 (1991)) and in natural human flora (Kroes, I., Lepp, P. W., Relman, D. A. Proc Nat Acad Sci (USA) 96, 14547-14552 (1999)). The drawback of this approach for unknown bioagent detection and epidemiology is that analysis of the PCR products requires the cloning and sequencing of hundreds to thousands of colonies per sample, which is impractical to perform rapidly or on a large number of samples.
Consensus priming has also been described for detection of several viral families, including coronaviruses (Stephensen, C. B., Casebolt, D. B. Gangopadhyay, N. N. Vir. Res. 60, 181-189 (1999)), enteroviruses (M. S. Oberste, K. Maher, M. A. Pallansch, J. Virol. 76, 1244-51 (2002); M. S. Oberste, W. A. Nix, K. Maher, M. A. Pallansch, J. Clin. Virol. 26, 375-7 (2003); M. S. Oberste, W. A. Nix, D. R. Kilpatrick, M. R. Flemister, M. A. Pallansch, Virus Res. 91, 241-8(2003)), retroid viruses (D. H. Mack, J. J. Sninsky, Proc. Natl. Acad. Sci. U.S.A. 85, 6977-81 (1988); W. Seifarth et al., AIDS Res. Hum. Retroviruses 16, 721-729 (2000); L. A. Donehower, R. C. Bohannon, R. J. Ford, R. A. Gibbs, J. Vir. Methods 28, 33-46 (1990)), and adenoviruses (M. Echavarria, M. Forman, J. Ticehurst, S. Dumler, P. Charache, J. Clin. Micro. 36, 3323-3326 (1998)). However, as with bacteria, there is no adequate analytical method other than sequencing to identify the viral bioagent present.
Methods of identification of bioagents are described in U.S. patent application Ser. No. 09/798,007, filed Mar. 3, 2001; Ser. No. 10/405,756, filed Mar. 31, 2003; Ser. No. 10/660,122, filed Sep. 11, 2003; and Ser. No. 10/728,486, filed Dec. 5, 2003, all of which are commonly owned and incorporated herein by reference in entirety as essential material.
Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. However, high-resolution MS alone fails to perform against unknown or bioengineered agents, or in environments where there is a high background level of bioagents (“cluttered” background). Low-resolution MS can fail to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to detect a particular organism.
Antibodies face more severe diversity limitations than arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones. Antibodies are only capable of detecting known agents in relatively uncluttered environments.
Several groups have described detection of PCR products using high resolution electrospray ionization—Fourier transform—ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS). Accurate measurement of exact mass combined with knowledge of the number of at least one nucleotide allowed calculation of the total base composition for PCR duplex products of approximately 100 base pairs. (Aaserud et al., J. Am. Soc. Mass Spec. 7:1266-1269, 1996; Muddiman et al., Anal. Chem. 69:1543-1549, 1997; Wunschel et al., Anal. Chem. 70:1203-1207, 1998; Muddiman et al., Rev. Anal. Chem. 17:1-68, 1998). Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS may be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al., Rapid Commun. Mass Spec. 10:377-382, 1996). The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for characterization of PCR products has been described. (Muddiman et al., Rapid Commun. Mass Spec. 13:1201-1204, 1999). However, the degradation of DNAs over about 75 nucleotides observed with MALDI limited the utility of this method.
U.S. Pat. No. 5,849,492 describes a method for retrieval of phylogenetically informative DNA sequences which comprise searching for a highly divergent segment of genomic DNA surrounded by two highly conserved segments, designing the universal primers for PCR amplification of the highly divergent region, amplifying the genomic DNA by PCR technique using universal primers, and then sequencing the gene to determine the identity of the organism.
U.S. Pat. No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods.
WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry.
WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single-stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand which is then analyzed by MS. Kits for target nucleic acid preparation are also provided.
PCT WO97/33000 discloses methods for detecting mutations in a target nucleic acid by nonrandomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by MS.
U.S. Pat. No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes.
WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry. Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation. Methods of sequencing a nucleic acid via mass spectrometry methods are also described.
WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry. U.S. Pat. Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing.
The present invention provides a novel approach for rapid, sensitive, and high-throughput identification of coronaviruses and includes the capability of identification of coronaviruses not yet observed and characterized. The methods described can be applied to additional viral families to cover a broad range of potential newly emerging viruses, or to bacterial, protozoal or fungal pathogens for epidemic disease surveillance in the future.